Expressão de genes relacionados ao metabolismo de nitrogênio, fósforo de potássio em cafeeiros submetidos ao estresse biótico.

As plantas freqüentemente são ameaçadas por agentes externos que podem ser fatores bióticos e/ou abióticos. Para cada tipo de ameaça há um requintado tipo de resposta que caracteriza o processo de defesa. Dentre os processos de defesa enquadram-se a ativação ou repressão de genes relacionados como quitinases, glucanaes, lipoxigenases, transferases, entre outros. Quaisquer modificações bioquímicas que ocorram durante os ataques dos patógenos são indispensáveis para a compreensão deste mecanismo biológico. Correlacionar os mecanismos biológicos de regulação e transporte de íons com os diferentes períodos de infestação e infecção é uma dessas maneiras de elucidar os processos que ocorrem durante a infestação e infecção. Neste estudo foram analisados os perfis de expressão de 5 genes (HAK-5, KEA, PAP-1, PII, NTR) relacionados com o transporte e regulação de potássio e nitrogênio com diferentes estímulos bióticos e em diferentes etapas de cada um desses processos. Os resultados demonstraram que há padrões diferenciais de regulação entre as plantas suscetíveis e resistentes para todos os genes analisados. Estas análises preliminares indicam que a regulação da absorção e/ou transporte de nutrientes exerce um importante papel durante as respostas de defesa em cafeeiros contra o bicho mineiro

Checagem da pureza genética de amostras de café bourbon por meio de marcador microssatélite.

O grupo Bourbon se destaca dentre as cultivares de café arábica pelas suas características sensoriais superiores e maior valorização no mercado. Além da origem, da sustentabilidade e dos aspectos físicos e sensoriais, a rastreabilidade dos cafés diferenciados é fundamental na valorização do produto final. Este trabalho avaliou a eficiência de um marcador de DNA do tipo microssatélite ou SSR, denominado LEG2, com o objetivo de diferenciar cultivares do grupo Bourbon das principais cultivares plantadas no Brasil. Após a identificação do polimorfismo, foram avaliados a repetibilidade da técnica, o perfil de amplificação em sementes e folhas e a análise de amostras de lavouras comerciais. Esse marcador diferencia o grupo Bourbon das demais cultivares IAC, à exceção de cultivares mais antigas e pouco plantadas no País, como Typica e Amarelo de Botucatu. As cultivares Tupi IAC 1669, do IAC, e Sabiá Tardio, da Fundação Procafé, também apresentam o mesmo alelo dos cafés do grupo Bourbon para o loco LEG2. Pela simplicidade e boa resolução da técnica, o SSR-LEG2 pode ser aplicado no controle de qualidade genética nas fases de pré e pós-melhoramento do cafeeiro, e também para checar a pureza genética de amostras comerciais de café Bourbon

Gene expression during response to nutrient deficiency in leaf-miner resistant and susceptible genotypes.

Several physiologic factors affect the defense response of coffee trees to the leaf-miner (Leucoptera coffeella), including nutrient availability during plant growth. Resistant coffee plants may exhibit leaf-miner infection when field nutritional status is critical. Knowledge of how biological mechanisms associated with transport of macronutrients and defense to leaf-miner infection interact may provide insights to elucidate the resistance response. In this study, we investigated the expression profile of genes from potassium and nitrogen metabolism, oxidative stress and defense response, in coffee seedlings submitted to limiting nutritional conditions. Young plants from both resistant and susceptible genotypes were irrigated with solutions containing variable macronutrients (N+K+, N+K-, NK+, NK-). After treatment, leaves were collected, total RNA extracted and gene expression was evaluated by qRT-PCR. All assessed genes exhibited differential expression between susceptible and resistant plants in response to nutritional stress. In addition, genes directly involved with defense mechanisms and oxidative stress exhibited the most significant expression differences when compared with genes from nutrient metabolism. These preliminary analyses suggest that regulation of nutrient up-take and/or transport may not play a central role during coffee response, and instead the activation of defense mechanisms is the initial response to low nutrient availabilit

Effects of somatic embryogenesis on gene expression of cloned coffee heterozygous hybrids.

Cloning of superior coffee plants by somatic embryogenesis can assist breeding programs on reducing the cost and time for launch of new cultivars. This study aimed to evaluate the efficiency of this methodology for cloning coffee trees with high heterozygosity, and to gather evidence that clonal progenies are faithful copies of mother plants. Selected plants IAC1 and IAC 2 from Coffea arabica breeding populations, resistant to leaf rust and leaf miner, respectively, were cloned via indirect somatic embryogenesis. Expression of selected genes involved in biological processes potentially affected by in vitro cultivation was evaluated by quantitative analysis. Genes encoding proteins associated with maintenance of DNA integrity and control of cell cycle presented predictable expression patterns along the clonal multiplication process. There were differences in the expression pattern of genes linked to in vitro cultivation-related stress, which were observed comparing either IAC 1 and IAC2 genotypes or clones and their corresponding mother plant. Those analyses suggest that the somatic embryogenesis does not lead to major genomic instability and clones are identical copies of mother plants, even with detected differences in the expression of genes that influence the response of in vitro cultivation

Caracterização molecular da interação cafeeiro/bicho-mineiro.

As plantas possuem sofisticados mecanismos de percepção à ataques externos e cujas informações são transmitidas de modo com que todo o seu sistema esteja preparado para agir em defesa própria. Para compreender as bases moleculares deste e outros mecanismos biológicos, pesquisas em larga escala envolvendo a expressão de genes são amplamente aplicadas. Para este estudo foram selecionados dois genótipos de cafeeiros, resistentes e suscetíveis ao bicho mineiro (Leucoptera coffeella), a fim de verificar as possíveis diferenças e semelhanças na expressão dos genes ao longo do processo de infestação. Para tanto, amostras de folhas das plantas foram coletadas em 3 tempos de infestação diferentes sendo eles, tempo zero (T0 ? antes da infestação), tempo 1 (T1 ? ovoposição) e tempo 2 (T2 ? eclosão dos ovos) e os RNA?s foram extraídos. A análise de microarranjo foi feita utilizando o modelo 12 X 135K e contou com um total de 22.500 sequencias de EST derivadas no banco de dados do genoma do café (CAFEST) onde cada seqüência foi representada por 6 oligos diferentes. As hibridizações, scanning e normalização dos dados foram realizados utilizando os programas Nimble Scan® e ArrayStar®. As expressões diferencias foram estabelecidas por comparação dos valores de fold change obtidos, e aquelas cujos valores ficaram +/- 2 foram consideradas não diferenciais. As análises de microarranjos indicaram que em todos os tempos testados houve uma pequena parcela do total de genes que apresentou expressão significativa. Nas plantas não infectadas houve expressão diferencial de 2137 genes (4,09% super expresso e 5,94% reprimidos em plantas suscetíveis). No tempo 1, os valores de fold change foram significativos para 2359 genes onde 5,29% foram super-expressos e 5,78 foram reprimidos em plantas suscetíveis. Em T2, 4,17% e 6,42% do total de genes foram super expressos e reprimidos respectivamente em plantas suscetíveis. Com os perfis obtidos foi possível selecionar genes que potencialmente podem distinguir uma interação compatível de uma incompatível e assim, estabelecer alguns dos genes responsáveis pelos mecanismos de defesa em cafeeiros contra o bicho-mineiro. Os genes selecionados servirão como base para buscas de polimorfismos gênicos que possam ser utilizados como marcadores para seleção assistida de progênies resistentes ao inseto

Large-scale analysis of differential gene expression in coffee genotypes resistant and susceptible to leaf miner-toward the identification of candidate genes for marker assisted-selection.

Background: A successful development of herbivorous insects into plant tissues depends on coordination of metabolic processes. Plants have evolved complex mechanisms to recognize such attacks, and to trigger a defense response. To understand the transcriptional basis of this response, we compare gene expression profiles of two coffee genotypes, susceptible and resistant to leaf miner (Leucoptera coffella). A total of 22000 EST sequences from the Coffee Genome Database were selected for a microarray analysis. Fluorescence probes were synthesized using mRNA from the infested and non-infested coffee plants. Array hybridization, scanning and data normalization were performed using Nimble Scan (R) e ArrayStar (R) platforms. Genes with foldchange values +/-2 were considered differentially expressed. A validation of 18 differentially expressed genes was performed in infected plants using qRT-PCR approach. Results: The microarray analysis indicated that resistant plants differ in gene expression profile. We identified relevant transcriptional changes in defense strategies before insect attack. Expression changes (>2.00-fold) were found in resistant plants for 2137 genes (1266 up-regulated and 873 down-regulated). Up-regulated genes include those responsible for defense mechanisms, hypersensitive response and genes involved with cellular function and maintenance. Also, our analyses indicated that differential expression profiles between resistant and susceptible genotypes are observed in the absence of leaf-miner, indicating that defense is already build up in resistant plants, as a priming mechanism. Validation of selected genes pointed to four selected genes as suitable candidates for markers in assisted-selection of novel cultivars. Conclusions: Our results show evidences that coffee defense responses against leaf-miner attack are balanced with other cellular functions. Also analyses suggest a major metabolic reconfiguration that highlights the complexity of this response

Effects of somatic embryogenesis on gene expression of cloned coffee heterozygous hybrids.

Cloning of superior coffee plants by somatic embryogenesis can assist breeding programs on reducing the cost and time for launch of new cultivars. This study aimed to evaluate the efficiency of this methodology for cloning coffee trees with high heterozygosity, and to gather evidence that clonal progenies are faithful copies of mother plants. Selected plants IAC1 and IAC 2 from Coffea arabica breeding populations, resistant to leaf rust and leaf miner, respectively, were cloned via indirect somatic embryogenesis. Expression of selected genes involved in biological processes potentially affected by in vitro cultivation was evaluated by quantitative analysis. Genes encoding proteins associated with maintenance of DNA integrity and control of cell cycle presented predictable expression patterns along the clonal multiplication process. There were differences in the expression pattern of genes linked to in vitro cultivation-related stress, which were observed comparing either IAC 1 and IAC2 genotypes or clones and their corresponding mother plant. Those analyses suggest that the somatic embryogenesis does not lead to major genomic instability and clones are identical copies of mother plants, even with detected differences in the expression of genes that influence the response of in vitro cultivation.Made available in DSpace on 2019-10-21T18:05:58Z (GMT). No. of bitstreams: 1 Effectsofsomaticembryogenesisongene.pdf: 969496 bytes, checksum: adf05ff04f9aef86bed834eda5415ea9 (MD5) Previous issue date: 2019bitstream/item/203223/1/Effects-of-somatic-embryogenesis-on-gene.pd